AMPK is a serine/threonine protein kinase involved in the regulation of cellular and organismal metabolism. The AMPK system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. AMPK has been suggested to serve as an energy gauge in cells, by detecting changes in the ratios of AMP to ATP. The consequences of AMPK activation include inhibition of ATP-consuming processes and activation of an ATP-producing process. The applicant's group was the first to demonstrate that physiologically relevant concentrations of ONOO- increased AMPK activity as well as its downstream enzymes such as eNOS and acetyl Co-A carboxylase (ACC). In addition, we have also demonstrated that ONOO--dependent AMPK activation is operative in hypoxia-reoxygenation, metformin- stimulated endothelial cells, thromboxane receptor stimulation, and nicotine-treated adipocytes. Similarly, we found that nicotine, a major constituent of cigarette smoke, activates AMPK in differentiated 3T3L1 adipocytes in a ROS-dependent fashion. Interestingly, AMPK activation by pharmacological AMPK activators (AICAR and metformin), physiological stimuli (glucose-depletion and osmotic stress), or genetic manipulation (adenoviruses encoding constitutively active AMPK) protects the endothelium against the adverse effects of nicotine. Concomitantly, pharmacological or genetic inhibition of AMPK markedly increased ROS, NF?B, and inflammatory genes (ICAM-1, VCAM-1, and E-selectin) in cultured endothelial cells, differentiated 3T3L1 adipocytes, and cultured primary rat hepatocytes. In parallel, in supporting that AMPK functions as a suppressor of oxidant stress, we have obtained preliminary evidence suggesting that AMPK activation by ischemic preconditioning (IPC) effectively blocked hypoxia/reoxygenation-triggered oxidant stress. The most conclusive evidence that AMPK reduced oxidant stress is that IPC failed to alter both the markers of oxidant stress and endothelial function in the AMPK 11 knockout (KO) mice. Consistently, we have found that compared to the wild types, aortas isolated from AMPK 11 or 12 KO mice exhibited impaired endothelial relaxation together with increased detections of both O2.- and ONOO-. Similarly, there was greater levels of NADPH oxidase subunits including gp91phox (NOX2), NOX-4, p22phox, p47phox, and p67phox together with increased NAD(P)H oxidase activity in AMPK12 KO mice than those in C57BL6 wild types. Importantly, apocynin, a potent NAD(P)H oxidase inhibitor, restored acetylcholine-induced endothelium-dependent relaxation in AMPK 12 KO mice, further suggesting NAD(P)H oxidase is functionally active and is responsible for impaired endothelial function in AMPK 12KO mice. Finally, without altering plasma lipids (cholesterol and triglyceride), the aortas isolated from Apo-E/AMPK11 or AMPK/AMPK 12 dual KO mice exhibited increased detection of oxidant stress markers, increased atherosclerotic lesions, and increased expression of proinflammatory adhesion molecules when compared to those in Apo-E KO mice. The goal of this application is to establish (a) that AMPK, through its ability to respond to very small changes in AMP levels, may be the proximal "oxidant stress-sensor" of the cell;(b) that AMPK activation may trigger physiological responses to suppress processes that generate oxidants (modulator) and or increase anti-oxidant defense systems;and (c) AMPK, via a reduction of oxidant stress, maintains the non-angiogenic, non-inflammatory, and atherosclerosis-resistant phenotypes in vascular cells. PUBLIC HEALTH RELEVANCE: A basic premise of this proposal is that AMPK activation could protect cells and the whole organism against the adverse effects of cardiovascular risk factors such as nicotine by setting in motion events that decrease oxidant stress. For the sake of clarity and being focused of the current application we use nicotine as a pathological stimulus relevant to cardiovascular diseases. As there is growing evidence that oxidant stress is involved in the vascular effects of cardiovascular risk factors including hypercholesterolemia, diabetes, and hypertension, thus, the significance of this proposal is beyond nicotine and will be applicable to other cardiovascular diseases. The current application addresses a fundamental question in biology and medicine, i.e., how oxidant stressors are sensed and modulated in health and disease. Completion of this application, thus, may add an entirely new dimension to the role of AMPK in stress responses and provide an interface between oxidant stress, energy metabolism, and cardiovascular biology. Completion of the proposed studies will provide novel insights into whether AMPK is a potential target for therapy in smoking and common diseases including aging, obesity, diabetes, hypertension, and atherosclerosis.